MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that function as key post-transcriptional regulators of gene expression in a range of eukaryotes, including humans. The small size of miRNA precursors, and the lack of antigenicity of miRNAs, makes them potentially ideal for viruses as inhibitors of innate or adaptive host cell defense pathways. Indeed, Epstein-Barr virus (EBV) was recently shown to encode several miRNAs and we have shown that Kaposi's sarcoma associated herpesvirus (KSHV) also expresses 11 distinct miRNAs in latently infected cells. This observation raises the possibility that these miRNAs may play an important role in KSHV replication and pathogenesis. In this application, we propose a series of experiments to define the cellular mRNA targets and biological functions of miRNAs encoded by KSHV, EBV and by the related Simian Gamma herpesviruses Rhesus Rhadinovirus (RRV) and herpesvirus Saimiri (HVS). In the case of KSHV and EBV, we will express individual or multiple KSHV miRNAs in uninfected human B cells and then measure the effect of miRNA expression on the mRNA expression profile, on the expression of specific cell surface makers and on the growth characteristics and apoptosis resistance of the cells. Similarly, we will block the expression of individual KSHV miRNAs in the latently KSHV infected cell line BCBL-1 using 2'-O-methyl antisense oligonucleotides and monitor not only the parameters listed above but also the effect on KSHV latency and activation of lytic replication in response to TPA treatment. For both HVS and RRV, we will first clone and characterize the miRNAs encoded by these viruses and then derive a series of RRV and HVS mutants lacking one or more of these miRNAs. The ability of these Simian viruses to replicate lytically or to establish latent infections will then be monitored in culture. Together, these studies should identify human mRNA targets for several KSHV and EBV miRNAs and shed light on the role of these miRNAs in the replication cycle of Gamma herpesviruses in general.